Insulin resistance is linked to many of the most prevalent and devastating age-related pathologies, including type 2 diabetes, cardiovascular disease and cognitive dysfunction. Skeletal muscle accounts for up to 85% of insulin-mediated blood glucose clearance, and glucose transport (GT) is a rate-controlling step for muscle glucose metabolism. Calorie restriction (CR) and exercise separately enhance insulin-mediated muscle GT in old rats, but the cellular mechanisms are poorly understood. Nothing is known about their combined effects on muscle insulin signaling or GT. The broad, long-term goal is to fully elucidate the independent and combined mechanisms for increased insulin sensitivity as the result of CR and/or exercise in older individuals. The Overall Hypothesis is: CR and exercise by old rats independently lead to increased insulin-stimulated glucose transport in skeletal muscle via distinct and overlapping mechanisms with further increases attained by the combination of CR and exercise. The Specific Aims are: 1) Determine in old rats the effects of CR and exercise, alone and in combination, on insulin-stimulated glucose transport and GLUT4 translocation in skeletal muscle; 2) Identify in old rats the specific insulin signaling events in skeletal muscle that are altered as the result of CR and exercise, alone and in combination; 3) Resolve in old rats which of the signaling events that are identified as altered by CR and/or exercise are responsible for greater insulin-stimulated GT with CR and exercise, alone and in combination; 4) Determine in old rats if differing shifts in lipids or glycogen in response to CR o exercise can act as triggers for the greater insulin-stimulated GT with CR and exercise, alone and in combination. Metabolic properties differ by muscle fiber type. Accordingly, muscle tissue (soleus and epitrochlearis, composed of primarily type I and type II fibers, respectively) and individually fiber-typed single fibers (GT of each fiber will be assayed by an innovative method) isolated from old rats after CR and/or exercise will be studied to discover processes that control muscle GT at both tissue and cell levels. Innovative genetic and pharmaceutical approaches will be used to elucidate mechanisms underlying CR and/or exercise benefits on GT in old rats. Greater levels of phosphorylated Akt2 (pAkt2) have been implicated as potentially pivotal for the CR-induced increase in GT. Recent data revealed the need to test if pAkt2 also contributes to greater insulin-mediated GT after exercise by older individuals. Muscle tissue and fibers from old rats after CR and/or exercise will be studied to determine: 1) novel mechanisms for enhanced pAkt2; 2) if greater pAkt2 is required for greater GT; 3) roles of established and newly identified Akt2 substrates in linking greater pAkt2 to increased GT; 4) possible Akt2-independent mechanisms for increases in GT; 5) the role of greater GLUT4 translocation in the elevated GT; and 6) unique roles of CR- and exercise-specific changes in lipids and glycogen as triggers for the CR and exercise effects on GT. The proposed research will provide novel insights into mechanisms for improved insulin sensitivity, a major health benefit of CR and/or exercise by older people.